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Half-calcified calmodulin promotes basal activity and inactivation of the L-type calcium channel Ca_V1.2.

ABSTRACT: The L-type Ca²⁺ channel Ca_V1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs Ca_V1.2 open probability (Po) and Ca²⁺-dependent inactivation (CDI) but the mechanisms remain unclear. Here, we present electrophysiological data that identify a half Ca²⁺-saturated CaM species (Ca₂/CaM) with Ca²⁺ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca²⁺ concentrations (50-100 nM) that constitutively preassociates with Ca_V1.2 to promote Po and CDI. We also present an NMR structure of a complex between the Ca_V1.2 IQ motif (residues 1644-1665) and Ca₂/CaM_12', a calmodulin mutant in which Ca²⁺ binding to EF1 and EF2 is completely disabled. We found that the CaM_12' N-lobe does not interact with the IQ motif. The CaM_12' C-lobe bound two Ca²⁺ ions and formed close contacts with IQ residues I1654 and Y1657. I1654A and Y1657D mutations impaired CaM binding, CDI, and Po, as did disabling Ca²⁺ binding to EF3 and EF4 in the CaM₃₄ mutant when compared to WT CaM. Accordingly, a previously unappreciated Ca₂/CaM species promotes Ca_V1.2 Po and CDI, identifying Ca₂/CaM as an important mediator of Ca signaling.

SUBMITTER: Bartels P

PROVIDER: S-EPMC9764201 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature

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Half-calcified calmodulin promotes basal activity and inactivation of the L-type calcium channel CaV1.2.

Bartels Peter P Salveson Ian I Coleman Andrea M AM Anderson David E DE Jeng Grace G Estrada-Tobar Zoila M ZM Man Kwun Nok Mimi KNM Yu Qinhong Q Kuzmenkina Elza E Nieves-Cintron Madeline M Navedo Manuel F MF Horne Mary C MC Hell Johannes W JW Ames James B JB

The Journal of biological chemistry 20221115 12

The L-type Ca2+ channel CaV1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs CaV1.2 open probability (Po) and Ca2+-dependent inactivation (CDI) but the mechanisms remain unclear. Here, we present electrophysiological data that identify a half Ca2+-saturated CaM species (Ca2/CaM) with Ca2+ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca2+< ...[more]

PMID: 36395884

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Project description:Pyridine nucleotides, such as NADPH and NADH, are emerging as critical players in the regulation of heart and vascular function. Glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme in the pentose phosphate pathway, is the primary source and regulator of cellular NADPH. In the current study, we have identified two isoforms of G6PD (slow and fast migrating) and functionally characterized the slow migrating isoform of G6PD (G6PD545) in bovine and human arteries. We found that G6PD545 is eluted in the caveolae fraction of vascular smooth muscle (VSM) and has a higher maximum rate of reaction (Vmax: 1.65-fold) than its fast migrating isoform (G6PD515). Interestingly, caveolae G6PD forms a complex with the pore-forming α1C-subunit of the L-type Ca2+ channel, Cav1.2, as demonstrated by a proximity ligation assay in fixed VSMCs. Additionally, Förster resonance energy transfer (FRET) analysis of HEK293-17T cells cotransfected with red fluorescent protein (RFP)-tagged G6PD545 (C-G6PD545) and green fluorescent protein (GFP)-tagged Cav1.2-(Cav1.2-GFP) demonstrated strong FRET signals as compared with cells cotransfected with Cav1.2-GFP and C-G6PD515. Furthermore, L-type Ca2+ channel conductance was larger and the voltage-independent component of availability (c1) was augmented in C-G6PD545 and Cav1.2-GFP cotransfectants compared with those expressing Cav1.2-GFP alone. Surprisingly, epiandrosterone, a G6PD inhibitor, disrupted the G6PD-Cav1.2 complex, also decreasing the amplitude of L-type Ca2+ currents and window currents, thereby reducing the availability of the c1 component. Moreover, overexpression of adeno-G6PD545-GFP augmented the KCl-induced contraction in coronary arteries compared with control. To determine whether overexpression of G6PD had any clinical implication, we investigated its activity in arteries from patients and rats with metabolic syndrome and found that G6PD activity was high in this disease condition. Interestingly, epiandrosterone treatment reduced elevated mean arterial blood pressure and peripheral vascular resistance in metabolic syndrome rats, suggesting that the increased activity of G6PD augmented vascular contraction and blood pressure in the metabolic syndrome. These data suggest that the novel G6PD-Cav1.2 interaction, in the caveolae fraction, reduces intrinsic voltage-dependent inactivation of the channel and contributes to regulate VSM L-type Ca2+ channel function and Ca2+ signaling, thereby playing a significant role in modulating vascular function in physiological/pathophysiological conditions.NEW & NOTEWORTHY In this study we have identified a novel isozyme of glucose-6-phosphate dehydrogenase (G6PD), a metabolic enzyme, that interacts with and contributes to regulate smooth muscle cell l-type Ca2+ ion channel function, which plays a crucial role in vascular function in physiology and pathophysiology. Furthermore, we demonstrate that expression and activity of this novel G6PD isoform are increased in arteries of individuals with metabolic syndrome and in inhibition of G6PD activity in rats of metabolic syndrome reduced blood pressure.

Project description:BackgroundL-type Ca2+ channel CaV1.2 is essential for cardiomyocyte excitation, contraction and gene transcription in the heart, and abnormal functions of cardiac CaV1.2 channels are presented in diabetic cardiomyopathy. However, the underlying mechanisms are largely unclear. The functions of CaV1.2 channels are subtly modulated by splicing factor-mediated alternative splicing (AS), but whether and how CaV1.2 channels are alternatively spliced in diabetic heart remains unknown.MethodsDiabetic rat models were established by using high-fat diet in combination with low dose streptozotocin. Cardiac function and morphology were assessed by echocardiography and HE staining, respectively. Isolated neonatal rat ventricular myocytes (NRVMs) were used as a cell-based model. Cardiac CaV1.2 channel functions were measured by whole-cell patch clamp, and intracellular Ca2+ concentration was monitored by using Fluo-4 AM.ResultsWe find that diabetic rats develop diastolic dysfunction and cardiac hypertrophy accompanied by an increased CaV1.2 channel with alternative exon 9* (CaV1.2E9*), but unchanged that with alternative exon 8/8a or exon 33. The splicing factor Rbfox2 expression is also increased in diabetic heart, presumably because of dominate-negative (DN) isoform. Unexpectedly, high glucose cannot induce the aberrant expressions of CaV1.2 exon 9* and Rbfox2. But glycated serum (GS), the mimic of advanced glycation end-products (AGEs), upregulates CaV1.2E9* channels proportion and downregulates Rbfox2 expression in NRVMs. By whole-cell patch clamp, we find GS application hyperpolarizes the current-voltage curve and window currents of cardiac CaV1.2 channels. Moreover, GS treatment raises K+-triggered intracellular Ca2+ concentration ([Ca2+]i), enlarges cell surface area of NRVMs and induces hypertrophic genes transcription. Consistently, siRNA-mediated knockdown of Rbfox2 in NRVMs upregulates CaV1.2E9* channel, shifts CaV1.2 window currents to hyperpolarization, increases [Ca2+]i and induces cardiomyocyte hypertrophy.ConclusionsAGEs, not glucose, dysregulates Rbfox2 which thereby increases CaV1.2E9* channels and hyperpolarizes channel window currents. These make the channels open at greater negative potentials and lead to increased [Ca2+]i in cardiomyocytes, and finally induce cardiomyocyte hypertrophy in diabetes. Our work elucidates the underlying mechanisms for CaV1.2 channel regulation in diabetic heart, and targeting Rbfox2 to reset the aberrantly spliced CaV1.2 channel might be a promising therapeutic approach in diabetes-induced cardiac hypertrophy.

Dataset Information

Half-calcified calmodulin promotes basal activity and inactivation of the L-type calcium channel Ca_V1.2.

Publications

Half-calcified calmodulin promotes basal activity and inactivation of the L-type calcium channel Ca<sub>V</sub>1.2.

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